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Published on: July 16, 2008
Michler's hydrol blue elucidates structural differences in prion strains
Yiling Xiao1, Sandra Rocha2, Catherine C Kitts3
1Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX 75390-8816.
Abstract:
Yeast prions provide self-templating protein-based mechanisms of inheritance whose conformational changes lead to the acquisition of diverse new phenotypes. The best studied of these is the prion domain (NM) of Sup35, which forms an amyloid that can adopt several distinct conformations (strains) that confer distinct phenotypes when introduced into cells that do not carry the prion. Classic dyes, such as thioflavin T and Congo red, exhibit large increases in fluorescence when bound to amyloids, but these dyes are not sensitive to local structural differences that distinguish amyloid strains. Here we describe the use of Michler's hydrol blue (MHB) to investigate fibrils formed by the weak and strong prion fibrils of Sup35NM and find that MHB differentiates between these two polymorphs. Quantum mechanical time-dependent density functional theory (TDDFT) calculations indicate that the fluorescence properties of amyloid-bound MHB can be correlated to the change of binding site polarity and that a tyrosine to phenylalanine substitution at a binding site could be detected. Through the use of site-specific mutants, we demonstrate that MHB is a site-specific environmentally sensitive probe that can provide structural details about amyloid fibrils and their polymorphs.
Insights
Michler's hydrol blue (MHB) is a novel fluorescent probe that distinguishes between different yeast prion (Sup35NM) amyloid strains. This environmentally sensitive dye offers new insights into amyloid fibril structures and polymorphs.
Area of Science:
- Biochemistry
- Structural Biology
- Yeast Genetics
Background:
- Yeast prions, like the Sup35NM protein, are self-templating entities that induce heritable phenotypic changes through conformational alterations.
- Amyloid fibrils formed by Sup35NM can exist in distinct conformational states (strains), each conferring unique cellular phenotypes.
- Conventional fluorescent dyes (e.g., thioflavin T) lack the sensitivity to differentiate between these amyloid strains based on subtle structural variations.
Purpose of the Study:
- To investigate the potential of Michler's hydrol blue (MHB) as a fluorescent probe for distinguishing between different amyloid polymorphs of the yeast prion Sup35NM.
- To elucidate the structural basis for MHB's differential fluorescence response to various amyloid conformations.
Main Methods:
- Synthesis and application of Michler's hydrol blue (MHB) for fluorescence analysis of Sup35NM amyloid fibrils.
- Utilizing site-specific mutants of Sup35NM to probe the binding interactions of MHB.
- Employing quantum mechanical time-dependent density functional theory (TDDFT) calculations to correlate MHB fluorescence with binding site polarity.
Main Results:
- MHB demonstrated the ability to differentiate between weak and strong prion fibril polymorphs of Sup35NM.
- TDDFT calculations revealed that MHB's fluorescence properties are sensitive to changes in binding site polarity.
- The probe could detect specific amino acid substitutions, such as tyrosine to phenylalanine, within the amyloid structure.
Conclusions:
- MHB serves as a site-specific, environmentally sensitive fluorescent probe for amyloid fibrils.
- This probe provides valuable structural details about amyloid fibrils and their distinct polymorphs, overcoming limitations of traditional dyes.
- MHB offers a promising tool for further research into prion structure-function relationships and amyloid diseases.
